Methods for using rose bengal for detection of chemical warfare agents

ABSTRACT

Rose Bengal for detecting a presence of chemical warfare agents. A method of detecting presence of a chemical warfare agent and includes applying a quinoid form of Rose Bengal to a substrate. When the substrate is exposed to the chemical warfare agent, a lactone form of Rose Bengal is spectrally observed because presence of the chemical warfare agent converts the quinoid form to the lactone form of Rose Bengal.

This application is a continuation of U.S. application Ser. Nos.14/520,545 and 14/520,569, filed Oct. 22, 2014, which claim the benefitof and priority to prior filed co-pending Provisional Application Ser.No. 61/894,112, filed 22 Oct. 2013. The application is also related toInternational Application No. PCT/GB2014/053,144, filed Oct. 21, 2014.The disclosure of each application is expressly incorporated herein byreference, in its entirety.

RIGHTS OF THE GOVERNMENT

The invention described herein may be manufactured and used by or forthe Government of the United States for all governmental purposeswithout the payment of any royalty.

FIELD OF THE INVENTION

The present invention relates generally to decontamination of substratesand assets and, more particularly, to methods and systems for detecting,decontaminating, and monitoring decontamination of substrates andassets.

BACKGROUND OF THE INVENTION

Traditional chemical warfare agent simulants and other problematiccontaminants, such as pesticides, are difficult to detect anddecontaminate, either by removal or decomposition. The U.S. Departmentof Defense has expended considerable effort in developing what arecalled “decontamination assurance sprays,” which indicate a presence ofcontamination, such as by a colorimetric change. However, theconventional decontamination assurance sprays do not decompose thecontaminants, nor do the decontamination assurance sprays provide anyinformation on when the contaminant is effectively removed orneutralized without reapplication.

Thus, there remains a need for decontamination assurance sprays that canremove or decompose a contaminant, provide feedback as to removaleffectiveness, or, preferably, both.

SUMMARY OF THE INVENTION

The present invention overcomes the foregoing problems and othershortcomings, drawbacks, and challenges of conventional decontaminationassurance sprays. While the invention will be described in connectionwith certain embodiments, it will be understood that the invention isnot limited to these embodiments. To the contrary, this inventionincludes all alternatives, modifications, and equivalents as may beincluded within the spirit and scope of the present invention.

A method of detecting presence of a chemical warfare agent and includesapplying a quinoid form of Rose Bengal to a substrate. When thesubstrate is exposed to the chemical warfare agent, a lactone form ofRose Bengal is spectrally observed because the presence of the chemicalwarfare agent converts the quinoid form to the lactone form of RoseBengal.

In accordance with some aspects of the present invention, a method ofdetermining presence of a chemical warfare agent includes applying aquinoid form of Rose Bengal to a substrate and spectrally observing theapplied Rose Bengal. Chemical warfare agent contamination is concludedif the observed spectrum corresponds to a lactone form of Rose Bengal.No chemical warfare agent contamination is concluded if the observedspectrum corresponds to the quinoid form of Rose Bengal.

Other embodiments of the present invention include a method applying aquinoid form of Rose Bengal to a substrate. The substrate is exposed toa nerve chemical warfare agent or a blister chemical warfare agent. Alactone form of Rose Bengal is spectrally observed because presence ofthe chemical warfare agent converts the quinoid form to the lactone formof Rose Bengal. A fluorescent signature of the exposed substrate isobserved.

Additional objects, advantages, and novel features of the invention willbe set forth in part in the description which follows and, in part, willbecome apparent to those skilled in the art upon examination of thefollowing or may be learned by practice of the invention. The objectsand advantages of the invention may be realized and attained by means ofthe instrumentalities and combinations particularly pointed out in theappended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate embodiments of the presentinvention and, together with a general description of the inventiongiven above, and the detailed description of the embodiments givenbelow, serve to explain the principles of the present invention.

FIGS. 1A and 1B are skeletal formulae of two isomer forms of RoseBengal, suitable for use with embodiments of the present invention.

FIG. 2 is the absorption spectrum of Rose Bengal in the presence ofdaylight (dashed line), ultraviolet radiation (solid line) and coolwhite light (dotted line).

FIG. 3 is the absorption spectrum of Rose Bengal over the visible lightspectrum for various concentrations of Rose Bengal (5 μM to 25 μM).

FIG. 4 is a flowchart illustrating a method of using Rose Bengal inaccordance with an embodiment of the present invention.

FIG. 5 is a bar graph illustrating a percent change in a concentrationof Demeton-S (illustrated as “[DEM]”) resulting from light exposure.

FIG. 6 is a skeletal formula illustrating a mechanism of Rose Bengalphotocatalytic oxidation of Demeton-S.

FIGS. 7A-7C are bar graphs illustrating percentage of end productsresulting from the Rose-Bengal included photocatalytic oxidation ofcontaminants at 10,000 LUX, 4,000 LUX, and Dark conditions,respectively.

FIGS. 8A-8F are chromatographs of end products of the photocatalyticoxidation of Demeton-S over a 24 hr residence time.

FIG. 9 is an exemplary Scanning Electron Microscopy image ofnanoparticles produced in accordance with an embodiment of the presentinvention.

FIGS. 10A-10F are spectra of 0.25 R560+1.00 RB+3.00 R640 nanoparticlesexposed to Demeton-S, diisopropyl fluorophosphates, andbis(2-chloroethyl) sulfide for 50 min with data collection every 10 min.

FIGS. 11A-11F are spectra of 0.50 R560+1.00 RB+3.00 R640 nanoparticlesexposed to Demeton-S, diisopropyl fluorophosphates, andbis(2-chloroethyl) sulfide for 50 min with data collection every 10 min.

FIGS. 12A-12F are spectra of 0.75 R560+1.00 RB+3.00 R640 nanoparticlesexposed to Demeton-S, diisopropyl fluorophosphates, andbis(2-chloroethyl) sulfide for 50 min with data collection every 10 min.

FIGS. 13A-13F are spectra of 1.00 R560+1.00 RB+1.00 R640 nanoparticlesexposed to Demeton-S, DFP and Mustard for 50 min with data collectionevery 10 min.

FIGS. 14A-14F are spectra of 1.00 R560+1.00 RB+2.00 R640 nanoparticlesexposed to Demeton-S, diisopropyl fluorophosphates, andbis(2-chloroethyl) sulfide for 50 min with data collection every 10 min.

FIGS. 15A-15F are spectra of 1.00 R560+1.00 RB+3.00 R640 nanoparticlesexposed to Demeton-S, diisopropyl fluorophosphates, andbis(2-chloroethyl) sulfide for 50 min with data collection every 10 min.

FIGS. 16A-16C are spectra of 1.00 RB nanoparticles exposed to Demeton-S,diisopropyl fluorophosphates, and bis(2-chloroethyl) sulfide,respectively, for 30 min with data collection every 10 min and with anexcitation wavelength of about 485 nm.

FIGS. 17A-17C are spectra of 2.00 RB nanoparticles exposed to Demeton-S,diisopropyl fluorophosphates, and bis(2-chloroethyl) sulfide,respectively for 30 min with data collection every 10 min and with anexcitation wavelength of about 485 nm.

FIGS. 18A-18C are spectra of 3.00 RB nanoparticles exposed to Demeton-S,diisopropyl fluorophosphates, and bis(2-chloroethyl) sulfide for 30 minwith data collection every 10 min and with an excitation wavelength ofabout 485 nm.

FIGS. 19A-19C are spectra of 4.00 RB nanoparticles exposed to Demeton-S,diisopropyl fluorophosphates, and bis(2-chloroethyl) sulfide for 30 minwith data collection every 10 min and with an excitation wavelength ofabout 485 nm.

FIGS. 20A-20C are spectra of 6.00 RB nanoparticles exposed to Demeton-S,diisopropyl fluorophosphates, and bis(2-chloroethyl) sulfide for 30 minwith data collection every 10 min and with an excitation wavelength ofabout 485 nm.

FIGS. 21A-21C are spectra of 1.00 RB against Demeton-S, diisopropylfluorophosphates, and bis(2-chloroethyl) sulfide, respectively.

FIGS. 22A-22C are spectra of 2.00 RB against Demeton-S, diisopropylfluorophosphates, and bis(2-chloroethyl) sulfide, respectively.

FIGS. 23A-23C are spectra of 3.00 RB against Demeton-S, diisopropylfluorophosphates, and bis(2-chloroethyl) sulfide, respectively.

FIGS. 24A-24C are spectra of 4.00 RB against Demeton-S, diisopropylfluorophosphates, and bis(2-chloroethyl) sulfide, respectively.

FIGS. 25A-25C are spectra of 6.00 RB against Demeton-S, diisopropylfluorophosphates, and bis(2-chloroethyl) sulfide, respectively.

FIGS. 26A-26C are spectra of 3.00 RB, 4.00 RB, and 6.00 RB,respectively, in a 1:2 dilution with Demeton-S.

FIGS. 27A-27C are spectra of 3.00 RB, 4.00 RB, and 6.00 RB,respectively, in a 1:5 dilution with Demeton-S.

FIGS. 28A-28C are spectra of 3.00 RB, 4.00 RB, and 6.00 RB,respectively, in a 1:10 dilution with Demeton-S.

FIGS. 29-29C are spectra of 2.00 RB, 4.00 RB, and 6.00 RB, respectively,in a 1:20 dilution with diisopropyl fluorophosphates.

FIGS. 30A-30C are spectra of 3.00 RB, 4.00 RB, and 6.00 RB,respectively, in a 1:30 dilution with diisopropyl fluorophosphates.

FIGS. 31A-31C are spectra of 3.00 RB, 4.00 RB, and 6.00 RB,respectively, in a 1:10 dilution with diisopropyl fluorophosphates.

FIGS. 32A-32C are spectra of 3.00 RB, 4.00 RB, and 6.00 RB,respectively, in a 1:20 dilution with bis(2-chloroethyl) sulfide.

FIGS. 33A-33C are spectra of 3.00 RB, 4.00 RB, and 6.00 RB,respectively, in a 1:40 dilution with bis(2-chloroethyl) sulfide.

FIGS. 34A-34C are spectra of 3.00 RB, 4.00 RB, and 6.00 RB,respectively, in a 1:60 dilution with bis(2-chloroethyl) sulfide.

FIGS. 35A-35C are spectra of 3.00 RB, 4.00 RB, and 6.00 RB,respectively, in a 1:40 dilution with bis(2-chloroethyl) sulfide.

FIGS. 36A-36C are spectra of 3.00 RB, 4.00 RB, and 6.00 RB,respectively, in a 1:160 dilution with bis(2-chloroethyl) sulfide.

It should be understood that the appended drawings are not necessarilyto scale, presenting a somewhat simplified representation of variousfeatures illustrative of the basic principles of the invention. Thespecific design features of the sequence of operations as disclosedherein, including, for example, specific dimensions, orientations,locations, and shapes of various illustrated components, will bedetermined in part by the particular intended application and useenvironment. Certain features of the illustrated embodiments have beenenlarged or distorted relative to others to facilitate visualization andclear understanding. In particular, thin features may be thickened, forexample, for clarity or illustration.

DETAILED DESCRIPTION OF THE INVENTION

Turning now to the figures, and in particular to FIGS. 1A and 1B, twoisomer forms of Rose Bengal (“RB”), a simple fluorescein analog, areshown. RB is generally non-toxic, relatively inexpensive,commercially-available, and FDA approved for, primarily medical,applications. RB further possesses unique chemical properties, describedin greater detail below, that neutralize some contaminants, such aschemical warfare agent simulants and pesticides, (collectively referredto as “contaminants”) while providing a mechanism (such asabsorption/color change and fluorescent emission characteristics) bywhich the presence of contaminants may be detected and decomposition ofthe contamination may be monitored. Moreover, RB shows strong dependenceof absorption and fluorescence spectra on pH such that spectra intensitydecreases with a drop in pH.

RB provides excellent fluorescence and absorbance (colorimetric)response to contaminants and, in the presence of light, effectively andefficiently decomposes the contaminants through a photocatalyticoxidation mechanism. In particular, the RB molecule consists of abenzene moiety, a xanthene moiety, and substituents that determine thephotochemical and physical properties. One derivative has thesubstituent R═NH(C₂H₅)₃ attached to a negatively charged oxygen as wellas to a carboxylic group to form a salt, which is referred to as aquinoid form (“q”) of RB and is shown in FIG. 1A. While not wishing tobe bound by theory, when q-RB is exposed to acidic environments, asprovided by most contaminants (including, chemical warfare agentsimulants, pesticides, and many toxic industrial chemicals), q-RBundergoes a conformational change from the quinoid form to a lactoneform (“l”), which is shown in FIG. 1B. When l-RB is exposed to alkalineconditions, the conformational change reverses to q-RB.

The conformational change between the isomer forms of FIGS. 1A and 1B isaccompanied by a visual, colorimetric change, wherein l-RB is visuallycolorless and q-RB is visually perceived as bright pink.

Both forms of RB are also known photocatalyst and, in the presence ofvisible light (absorption spectrum is shown in FIG. 2), converts ambienttriplet state oxygen to the more active and oxidative singlet state,which is a known decontaminant for a number of contaminants.

With reference now to FIG. 4, and in use in accordance with anembodiment of the present invention, RB may be applied to a substrate orasset (Block 100) after or before exposing the substrate or asset to atleast one contaminant in order to detect the contaminant, and to monitorits decomposition (Blocks 102, 104, respectively). The exposure need notbe intentional.

RB may be applied neat, such as an additive to aqueous- or solvent-basedsystems and for application to contaminated substrate or asset.According to another embodiment, RB may be cross-linked into fabrics,polymers, or coatings at least partially comprising the substrate orasset and via established crosslinking methods for contaminationdetection and self-decontamination. In still other embodiments, RB maybe cross-linked into nanoparticles for industrial or remediationapplications. Suitable cross-linking methods and mechanisms are known bythe skilled artisan and may include, for example, thermal attachments,microwave attachment, physical adsorption, polymeric attachment, orcross-linking agents (such as acrylates, silanes, epoxides, vinylgroups, and so forth). Cross-linking to nanoparticles may alternativelybe accomplished according to the methods taught in U.S. ProvisionalApplication No. 61/829,557, filed May 31, 2013, and entitled CONTROLLEDMICROWAVE ASSISTED-SYNTHESIS OF FUNCTIONALIZED SILICA NANOPARTICLES;International Application No. PCT/GB2014/051644, filed May 29, 2014, andentitled CONTROLLED MICROWAVE ASSISTED SYNTHESIS OF FUNCTIONALIZEDSILICA NANOPARTICLES; and U.S. Non-Provisional application Ser. No.14/290,336, filed May 29, 2014, and entitled CONTROLLED MICROWAVEASSISTED SYNTHESIS OF FUNCTIONALIZED SILICA NANOPARTICLES. Thedisclosure of each application is incorporated herein by reference, inits entirety. Due to its high solubility in water, RB may alternativelybe bind to a porous surface of the nanoparticle thoughfunctionalization, encapsulation, or trapping dye molecules.Functionalization or trapping may prevent dilution of dye molecules inwater or water-based solutions and subsequent escape of the moleculesfrom the surface upon removal of water.

According to yet other embodiments, RB may be directly integrated into acoating or into fluids to provide chemical warfare agent simulantdetection, decontamination, and decontamination assurance sprays.

In Block 106, the contaminated substrate or asset may then be exposed toradiation having a wavelength ranging from 400 nm to 700 nm fordetection of at least contaminant. For purposes of decontamination,exposure to light may continue, while monitoring a fluorescentsignature, absorbance signature, or both, of the substrate or assetunder exposure to radiation (“Yes” branch of Decision Block 108).Otherwise, if monitoring for detection, contamination, or both iscomplete (“No” branch of Decision Block 108), then exposing thesubstrate or asset to radiation may be terminated (Block 110) and theprocess ends.

Use of RB may also include, according to some embodiments of the presentinvention, additional dyes for additional, enhanced, or alternativedetections.

The following examples illustrate particular properties and advantagesof some of the embodiments of the present invention. Furthermore, theseare examples of reduction to practice of the present invention andconfirmation that the principles described in the present invention aretherefore valid but should not be construed as in any way limiting thescope of the invention.

EXAMPLE 1

RB was purchased in its pure form (Pfaltz & Bauer, Waterbury, Conn.) andadded in 0.5 wt. %, 1.0 wt. %, 2.0 wt. %, and 5.0 wt. % loadings tocommercially-available, MIL-PRF-85285 compliant, aerospace coatings(obtained from PPG Industries, Irvine, Calif.) and tested againstchemical warfare agent simulants under simulated light and darkconditions.

As shown in FIG. 3, all compositions containing RB demonstrated highlevels of agent and simulant decomposition. In FIG. 5, changes in apercent concentration of DEM resulting from light and dark conditionsare shown as bar graphs for each coating tested.

EXAMPLE 2

The coatings of Example 1 were subjected to 4 g/m² Demeton-S (“DEM”) for24 hr in complete darkness, simulated indirect sun, and simulated directsun conditions.

FIG. 6 illustrates a RB-based photocatalytic oxidation mechanism ofDemeton-S 112 into an elimination product and while FIGS. 7A-7C aregraphical representations of data obtained when coatings having RB asdescribed above and exposed to DEM are irradiated at 10,000 LUX, 4,000LUX, and Dark conditions, respectively. Each graph illustrates arelative percentage of unreacted Demeton-S (“% DEM”), the eliminationproduct (“% ELIM”), and the neutralized Demeton-S Sulfone (“% SULF”).

FIGS. 8A-8F are graphical representations of chromatographs of the 24 hrresidence time photoactivity versus Demeton-S. The internal standard,tetralin, is the predominate peak appearing at about 3.85 hr. Moreparticularly, FIG. 8A is a graphical representation of the chromatographof 1.0 wt. % RB photocatalytic oxidation of DEM in MIL-PRF-85285 paint(obtained from PPG Industries) under dark conditions; FIG. 8B agraphical representation of the chromatograph of 1.0 wt. % RBphotocatalytic oxidation of DEM in MIL-PRF-85285 under indirect light(4,000 LUX) conditions; and FIG. 8C a graphical representation of thechromatograph of 1.0 wt. % RB photocatalytic oxidation of DEM inMIL-PRF-85285 under direct light (10,000 LUX) conditions.

FIGS. 8D-8F are similar to FIGS. 8A-8C, but demonstrate synergisticeffects of 0.5 wt. % RB with 0.5 wt. % 1,2-benzisothiaole-3(2H)-one(“BIT”).

EXAMPLE 3

RB was cross-linked into silica nanoparticles fabricated in accordancewith the methods described in U.S. Provisional Application No.61/829,557; International Application No. PCT/GB2014/051644; U.S.Non-Provisional application Ser. No. 14/290,336, which are discussedabove. One particular method is described in detail below. Subsequently,the RB-cross-linked silica nanoparticles were cross-linked onto cottonfibers using microwave assisted synthesis methods. RB was also combinedwith at least one secondary dye, Rhodamine 560 (“R560”), Rhodamine 640(“R640”), or both, which were also cross-linked into silicananoparticles and cotton fibers employing similar methods. The silicananoparticles were tested using absorbance and fluorescent spectra inthe presence of each of three chemical agent simulants: Demeton-S,diisopropyl fluorophosphates (“DFP”), and bis(2-chloroethyl) sulfide(“Mustard,” or otherwise known to those skilled in the art as “sulfurmustard”).

According to one example, 0.7 mL of H₂O is mixed with 1 mL of HCl and 1mL of tetraethyl orthosilicate (“TEOS”). The solution was mixed for 30sec. X mg (0.25 mg to 0.75 mg) of R560, 1.00 mg of RB, and Z mg (1.00 mgto 3.00 mg) of R640 were added to 40 mL of acetone and mixed. 0.35 mL ofthe hydrolyzed TEOS solution were added to the dye solution and mixedfor 30 sec. 5 mL of the final solution were placed in a 10 mL CEM vialand subjected to microwave field (300 W) until the surface of the vialhas reached 125° C., which was then maintained for 60 sec.

Diameters of resultant particles ranged from about 200 nm to about 300nm, as measured by a scanning electron microscope (“SEM”) (S-2600N,Hitachi, Ltd., Tokyo, Japan) and dynamic light scattering (“DLS”)(Nano-ZS90, Malvern Instruments Ltd., Worcestershire, UK). An exemplarySEM image of the resultant nanoparticles is shown in FIG. 9.

EXAMPLE 4

A 100 μL suspension of nanoparticles formed according to the methoddescribed in Example 3 was injected into each well of a 96-well plateand mixed with 200 μL of water. 1 μL of a contaminant (either Demeton-Sor DFP) was carefully placed on top of the suspension surface withoutmixing. Time dependent spectra were collected using a plate reader(BioTek Synergy™ 4 Hybrid Microplate Reader (BioTek Instruments, Inc.,Winooski, Vt.)) using 2 excitation wavelengths: 450 nm and 485 nm. Eachrun was 50 min long with 10 min time intervals between measurements.Each plot was normalized with respect to a corresponding referencesample.

0.25 R560+1.00 RB+3.00 R640: FIGS. 10A-10F are spectra from a samplecomprising 0.25 R560+1.00 RB+3.00 R640 nanoparticles and exposed toDemeton-S, DFP, and Mustard for 50 min with data collection every 10min. FIGS. 10A and 10B illustrate results versus Demeton-S at 450 nm and485 nm, respectively; FIGS. 10C and 10D illustrate results versus DFP at450 nm and 485 nm, respectively; and FIGS. 10E and 10F illustrateresults versus Mustard at 450 nm and 485 nm, respectively. These datademonstrate that the nanoparticles respond to the presence of simulantsfor both excitation wavelengths. The spectra produced by excitation at485 nm shows similar response to each of the three simulants withstabilization time at about 20 min.

0.50 R560+1.00 RB+3.00 R640: FIGS. 11A-11F are spectra of a samplecomprising 0.50 R560+1.00 RB+3.00 R640 nanoparticles and exposed toDemeton-S, DFP, and Mustard for 50 min with data collection every 10min. FIGS. 11A and 11B illustrate results versus Demeton-S at 450 nm and485 nm, respectively; FIGS. 11C and 11D illustrate results versus DFP at450 nm and 485 nm, respectively; and FIGS. 11E and 11F illustrateresults versus Mustard at 450 nm and 485 nm, respectively. The 0.50R560+RB+3 R640 nanoparticles show responses (FIGS. 11A-11F) similar theresponses of the 0.25 R560+1.00 RB+3.00 R640 nanoparticles (FIG.10A-10F); however, the change in fluorescent spectra is not as dramaticexhibiting equilibration time closer to 10 min rather than 20 min as inthe case of the 0.25 R560+1.00 RB+3.00 R640 nanoparticles.

0.75 R560+1.00 RB+3.00 R640: FIGS. 12A-12F are spectra of a samplecomprising 0.75 R560+1.00 RB+3.00 R640 nanoparticles and exposed toDemeton-S, DFP, and Mustard for 50 min with data collection every 10min. FIGS. 12A and 12B illustrate results versus Demeton-S at 450 nm and485 nm, respectively; FIGS. 12C and 12D illustrate results versus DFP at450 nm and 485 nm, respectively; and FIGS. 12E and 12F illustrateresults versus Mustard at 450 nm and 485 nm, respectively. The 0.75R560+1.00 RB+3.00 R640 nanoparticles show even less dramatic change influorescence spectra with exception of Mustard. The 0.75 R560+1.00RB+3.00 R640 nanoparticles response time was under 10 min with dramaticchange in intensity of the spectra.

1.00 R560+1.00 RB+1.00 R640: FIGS. 13A-13F are spectra of a samplecomprising 1.00 R560+1.00 RB+1.00 R640 nanoparticles and exposed toDemeton S, DFP, and Mustard for 50 min with data collection every 10min. FIGS. 13A and 13B illustrate results versus Demeton-S at 450 nm and485 nm, respectively; FIGS. 13C and 13D illustrate results versus DFP at450 nm and 485 nm, respectively; and FIGS. 13E and 13F illustrateresults versus Mustard at 450 nm and 485 nm, respectively. TheR560+RB+R640 nanoparticles show good response for DFP regardless of theexcitation wavelength; however, a response to Mustard takes under 20 minwith 450 nm exposure and 50 min with 485 nm exposure.

1.00 R560+1.00 RB+2.00 R640: FIGS. 14A-14F are spectra of a samplecomprising 1.00 R560+1.00 RB+2.00 R640 nanoparticles and exposed toDemeton-S, DFP, and Mustard for 50 min with data collection every 10min. FIGS. 14A and 14B illustrate results versus Demeton-S at 450 nm and485 nm, respectively; FIGS. 14C and 14D illustrate results versus DFP at450 nm and 485 nm, respectively; and FIGS. 14E and 14F illustrateresults versus Mustard at 450 nm and 485 nm, respectively.

1.00 R560+1.00 RB+3.00 R640: FIGS. 15A-15F are spectra of 1.00 R560+1.00RB+3.00 R640 nanoparticles and exposed to Demeton-S, DFP, and Mustardfor 50 min with data collection every 10 min. FIGS. 15A and 15Billustrate results versus Demeton-S at 450 nm and 485 nm, respectively;FIGS. 15C and 135 illustrate results versus DFP at 450 nm and 485 nm,respectively; and FIGS. 15E and 15F illustrate results versus Mustard at450 nm and 485 nm, respectively. The 1.00 R560+1.00 RB+3.00 R640nanoparticles show significant response if excited at 485 nm for allthree simulants, exhibiting stabilization times under 10 min.

EXAMPLE 5

1.00 RB: FIGS. 16A-16C are spectra of a sample comprising 1.00 RBnanoparticles exposed to Demeton-S, DFP, and Mustard, respectively, for30 min with data collection every 10 min and with an excitationwavelength of about 485 nm. The single dye nanoparticles were exposedonly to 485 nm excitation wavelength due to very weak light absorptionat 450 nm. The 1.00 RB nanoparticles demonstrated a response to DFP(FIG. 16B) and Mustard (FIG. 16C) with stabilization times ranging from10 min to 20 min respectively. Demeton-S (FIG. 16A) caused an initialrise in the spectrum upon addition of the chemical warfare agentsimulant, followed by a decrease of the intensity to below the referenceline.

2.00 RB: FIGS. 17A-17C are spectra of a sample comprising 2.00 RBnanoparticles exposed to Demeton-S, DFP, and Mustard, respectively for30 min with data collection every 10 min and with an excitationwavelength of about 485 nm. The 2.00 RB nanoparticles demonstrate an“oscillating” behavior of the spectra, with an initial increase abovereference line followed by a drop below the reference line.

3.00 RB: FIGS. 18A-18C are spectra of a sample comprising 3.00 RBnanoparticles exposed to Demeton-S, DFP, and Mustard for 30 min withdata collection every 10 min and with an excitation wavelength of about485 nm. The 3.00 RB nanoparticles were very responsive to Demeton-S(FIG. 18A) and DFP (FIG. 18B) while the 3.00 RB nanoparticles withMustard (FIG. 18C) had spectra coinciding with reference spectra after30 min. The presence of DFP caused the fluorescence peak to split.

4.00 RB: FIGS. 19A-19C are spectra of a sample comprising 4.00 RBnanoparticles exposed to Demeton-S, DFP, and Mustard for 30 min withdata collection every 10 min and with an excitation wavelength of about485 nm. The 4.00 RB nanoparticles were very responsive to Demeton-S(FIG. 19A) and DFP (FIG. 19B) while the 4.00 RB nanoparticles withMustard (FIG. 19C) had spectra coinciding with reference spectra after30 min. The presence of DFP caused the fluorescence peak to split.

6.00 RB: FIGS. 20A-20C are spectra of a sample comprising 6.00 RBnanoparticles exposed to Demeton-S, DFP, and Mustard for 30 min withdata collection every 10 min and with an excitation wavelength of about485 nm. The 6.00 RB nanoparticles were very responsive to Demeton-S(FIG. 20A) and DFP (FIG. 20B) while the 6.00 RB nanoparticles withMustard (FIG. 20C) had spectra coinciding with reference spectra after30 min. The presence of any of one of the chemical warfare agentsimulants caused the fluorescence peak to split.

EXAMPLE 6

X RB: FIGS. 21A-25C demonstrate a high response with fast stabilizationtime of under 10 min (except for Demeton-S) for X RB nanoparticles,wherein X ranges from 1.00 to 6.00.

FIGS. 21A-21C are spectra of 1.00 RB against Demeton-S, DFP, andMustard, respectively.

FIGS. 22A-22C are spectra of 2.00 RB against Demeton-S, DFP, andMustard, respectively.

FIGS. 23A-23C are spectra of 3.00 RB against Demeton-S, DFP, andMustard, respectively.

FIGS. 24A-24C are spectra of 4.00 RB against Demeton-S, DFP, andMustard, respectively.

FIGS. 25A-25C are spectra of 6.00 RB against Demeton-S, DFP, andMustard, respectively.

All samples responded to the presence of simulants and have faststabilization time under 10 min.

EXAMPLE 7

X RB dilutions: FIGS. 26A-36C demonstrate the response of RB versusvarious dilutions with each chemical warfare agent simulant, wherein Xranges from 3.00 to 6.00.

100 μL aliquots of samples each comprising one of 3.00 RB nanoparticles,4.00 RB nanoparticles, and 6.00 RB nanoparticles were mixed with 200 μLof water and either 0.5 μL or 0.25 μL (i.e., 1-to-2 and 1-to-5dilutions, respectively) of Demeton-S. FIGS. 26A-26C are spectra of 3.00RB, 4.00 RB, and 6.00 RB, respectively, in a 1:2 dilution withDemeton-S; FIGS. 27A-27C are spectra of 3.00 RB, 4.00 RB, and 6.00 RB,respectively, in a 1:5 dilution with Demeton-S; and FIGS. 28A-28C arespectra of 3.00 RB, 4.00 RB, and 6.00 RB, respectively, in a 1:10dilution with DFP. A detection limit of 0.5 μL per 300 μL (100 μL ofeach sample with 200 μL of water) was determined.

100 μL of samples each comprising one of 2.00 RB nanoparticles, 3.00 RBnanoparticles, 4.00 RB nanoparticles, and 6.00 RB nanoparticles weremixed with 200 μL of water and 0.1 μL, 0.05 μL, or 0.03 μL of DFP. FIGS.29A-29C are spectra of 2.00 RB, 4.00 RB, and 6.00 RB, respectively, in a1:20 dilution with DFP; FIGS. 30A-30C are spectra of 3.00 RB, 4.00 RB,and 6.00 RB, respectively, in a 1:30 dilution with DFP; and FIGS.31A-31C are spectra of 3.00 RB, 4.00 RB, and 6.00 RB, respectively, in a1:10 dilution with DFP.

100 μL of samples each comprising one of 3.00 mg RB nanoparticles, 4.00mg RB nanoparticles, and 6.00 mg of RB nanoparticles sample were mixedwith 200 μL of water and 0.1 μL, 0.05 μL, or 0.03 μL of Mustard. FIGS.32A-32C are spectra of 3.00 RB, 4.00 RB, and 6.00 RB, respectively, in a1:20 dilution with Mustard; FIGS. 33A-33C are spectra of 3.00 RB, 4.00RB, and 6.00 RB, respectively, in a 1:40 dilution with Mustard; FIGS.34A-34C are spectra of 3.00 RB, 4.00 RB, and 6.00 RB, respectively, in a1:60 dilution with Mustard; FIGS. 35A-35C are spectra of 3.00 RB, 4.00RB, and 6.00 RB, respectively, in a 1:40 dilution with Mustard; andFIGS. 36A-36C are spectra of 3.00 RB, 4.00 RB, and 6.00 RB,respectively, in a 1:160 dilution with Mustard. A detection limit as lowas 0.006 μL per 300 μL (100 μL of sample with 200 μL of water) forMustard was determined.

While the present invention has been illustrated by a description of oneor more embodiments thereof and while these embodiments have beendescribed in considerable detail, they are not intended to restrict orin any way limit the scope of the appended claims to such detail.Additional advantages and modifications will readily appear to thoseskilled in the art. The invention in its broader aspects is thereforenot limited to the specific details, representative apparatus andmethod, and illustrative examples shown and described. Accordingly,departures may be made from such details without departing from thescope of the general inventive concept.

What is claimed is:
 1. A method of detecting presence of a chemicalwarfare agent, the method comprising: applying a quinoid form of RoseBengal to a substrate; exposing the substrate to a chemical warfareagent; and spectrally observing a lactone form of Rose Bengal, whereinthe chemical warfare agent converts the quinoid form to the lactone formof Rose Bengal.
 2. The method of claim 1, further comprising: observinga fluorescent signature of the exposed substrate, wherein presence offluorescence within the fluorescent signature indicates the lactone formof Rose Bengal.
 3. The method of claim 1, wherein applying the quinoidform of Rose Bengal further comprises: applying a secondary dye.
 4. Themethod of claim 3, wherein the secondary dye is Rhodamine 560, Rhodamine640, or both.
 5. The method of claim 1, wherein applying the quinoidform of Rose Bengal further comprises: binding the quinoid form of RoseBengal to the substrate.
 6. The method of claim 5, wherein binding thequinoid form of Rose Bengal includes functionalization, encapsulation,or trapping.
 7. The method of claim 1, wherein applying the quinoid formof Rose Bengal further comprises: binding the quinoid form of RoseBengal to nanoparticles; and cross-linking the nanoparticles to thesubstrate.
 8. The method of claim 1, further comprising: decomposing thechemical warfare agent by a Rose Bengal induced photocatalytic oxidationmechanism.
 9. The method of claim 8, wherein decomposing the chemicalwarfare agent further comprises: a light having a wavelength within thevisible spectrum.
 10. A method of determining presence of a chemicalwarfare agent, the method comprising: applying a quinoid form of RoseBengal to a substrate; spectrally observing the applied Rose Bengal, andconcluding chemical warfare agent contamination if the observed spectrumcorresponds to a lactone form of Rose Bengal or concluding no chemicalwarfare agent contamination if the observed spectrum corresponds to thequinoid form of Rose Bengal.
 11. The method of claim 10, furthercomprising: spectrally observing a fluorescence signature; andconcluding chemical warfare agent contamination if fluorescence isobserved in the fluorescence signature or concluding no chemical warfareagent contamination fluorescence is observed in the fluorescencesignature.
 12. The method of claim 10, wherein applying the quinoid formof Rose Bengal further comprises: applying a secondary dye.
 13. Themethod of claim 10, wherein the secondary dye is Rhodamine 560,Rhodamine 640, or both.
 14. The method of claim 13, wherein applying thequinoid form of Rose Bengal further comprises: binding the quinoid formof Rose Bengal to the substrate.
 15. The method of claim 14, whereinbinding the quinoid form of Rose Bengal includes functionalization,encapsulation, or trapping.
 16. The method of claim 10, wherein applyingthe quinoid form of Rose Bengal further comprises: binding the quinoidform of Rose Bengal to nanoparticles; and cross-linking thenanoparticles to the substrate.
 17. The method of claim 10, furthercomprising: decomposing the chemical warfare agent by a Rose Bengalinduced photocatalytic oxidation mechanism.
 18. The method of claim 16,wherein decomposing the chemical warfare agent further comprises: alight having a wavelength within the visible spectrum.
 19. A method ofdetecting presence of a chemical warfare agent, the method comprising:applying a quinoid form of Rose Bengal to a substrate; exposing thesubstrate to a nerve chemical warfare agent or a blister chemicalwarfare agent; spectrally observing a lactone form of Rose Bengal; andobserving a fluorescent signature of the exposed substrate.
 20. Themethod of claim 19, wherein applying the quinoid form of Rose Bengalfurther comprises: binding the quinoid form of Rose Bengal to thesubstrate.
 21. The method of claim 19, wherein the nerve chemicalwarfare agent is propan-2-yl methylphosphonofluoridate.
 22. The methodof claim 19, wherein the blister chemical warfare agent isbis(2-chloroethyl)sulfide, diisopropyl fluorophosphates, Demeton-S, oranother phosphate ester.